Antibody for binding to interleukin 4 receptor

ABSTRACT

Disclosed is an antibody capable of binding to the interleukin 4 (IL-4) receptor (IL-4). Also disclosed are a nucleic acid sequence encoding the antibody, a vector including the nucleic acid sequence, and a host cell transformed or transfected with the vector. Provided are a method for producing the antibody, a medical use of the antibody, and a kit including the antibody.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.16/809,411, filed Mar. 4, 2020, now U.S. Pat. No. 10,774,141, issuedSep. 15, 2020, which is a continuation application of U.S. applicationSer. No. 16/307,930, filed Dec. 6, 2018 (abandoned), which is theNational Stage of International Application Number PCT/CN2017/087592,filed on Jun. 8, 2017, which claims priority of Chinese PatentApplication Number 201610399254.4, filed on Jun. 8, 2016, the entirecontents of each of which are incorporated herein by reference.

INCORPORATION BY REFERENCE

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy was modified on Aug. 14,2020, is named SUZ-001C1D1_Sequence_Listing.txt, and is 71,361 bytes insize.

TECHNICAL FIELD

The present invention relates to the field of biopharmaceutics.Particularly, the present invention relates to an antibody that iscapable of binding to interleukin 4 (IL-4) receptor (IL-4R) and usesthereof.

BACKGROUND OF THE INVENTION

Interleukin-4 (IL-4) is a cytokine produced primarily by activated Tcells, monocytes, basophils, mast cells, and eosinophils. IL-4 isinvolved in a variety of biological processes, and its biologicaleffects known in the art include stimulating the proliferation ofactivated B cells and T cells and the differentiation of CD4+ T cellsinto type II helper T cells. What's more, studies have shown that IL-4has multiple effects in mediating immune responses to diseases such asallergic diseases, autoimmune diseases, infectious diseases and tumors,and has therapeutic effects on tumors, autoimmune diseases andinfectious diseases and the like. Meanwhile, IL-4 can also regulateimmune response to vaccine. Therefore, IL-4 has always been one hot areafor research attracting extensive attentions.

IL-13 is also a cytokine produced by activated T cells, which hasdifferent functions in different types of cells, such as monocytes, Bcells, mast cells and keratinocytes. IL-13 can inhibit the release ofinflammatory cytokines and chemokines from monocytes, induce theproliferation and differentiation of B cells, and promote the synthesisof IgE. IL-13 and IL-4 share many common properties in terms ofbiological functions, including inhibiting the release of inflammatorymediators from monocytes, inducing dendritic-like development ofmacrophages, promoting the expression of CD23 on the surface ofmonocytes and stimulating the synthesis of immunoglobulins by B cells.At the same time, IL-13 also has its own biological features, mainlyincluding: promoting the differentiation of human monocytes and changesof antigens on cell surface; inducing the proliferation anddifferentiation of B cells, and promoting the secretion of antibodiesfrom B cells; regulating the synthesis of IgE, thereby being associatedwith allergic reactions in the body; inhibiting the growth of tumorcells; inhibiting the replication of HIV; and the like.

The biological activity of IL-4 is mediated by a specific IL-4 receptoron cell surface (IL-4R, which is called “hIL-4R” in human). Human IL-4Ris a heterodimer formed by two polypeptide chains, in which the alphachain (hIL-4Rα, UniProtKB: P24394) has a high affinity for IL-4. Andstudies have shown that the cell surface receptor alpha chain of IL-13(IL-13Rα chain) also forms another form of IL-4R complex with IL-4Rαchain. Since the IL-4Rα chain in the IL-4R complex plays a leading rolein binding to IL-4 and other cytokines are involved, the IL-4Rα chain iscurrently being studied as a major target. What's more, human monoclonalantibodies against the IL-4Rα chain have been clinically proven to beeffective in relieving and treating conditions such as asthma, eczema,atopic dermatitis and the like.

Human interleukin-4 receptor is known to produce a soluble form ofprotein (shIL-4Rα, SEQ ID NO: 94) that inhibits cell proliferationmediated by IL-4 and IL-5 up-regulation mediated by T cells. Two formsof the receptor are associated with allergic reaction, which manifestsas diseases like allergic rhinitis, sinusitis, asthma, eczema and so on.Therefore, blocking antibodies that target the protein can help treatand relieve said diseases.

Asthma is a chronic airway inflammatory disease, in which manyinflammatory cells, such as eosinophils, mast cells and lymphocytes areinvolved, and its specific pathogenesis is still unclear. Sincecytokines such as IL-4 play an important role in the occurrence anddevelopment of bronchial asthma, the development of antibodies specificfor IL-4 is one of the effective ways to treat asthma. Inhibition ofIL-4/IL-4Rα can have an effective immunomodulatory effect on asthma.

Allergic rhinitis (AR) has a pathogenesis much in common with that ofasthma, and it and asthma both belong to type I allergy. Meanwhile,studies have found that IL-4, IL-17 and IgE play an important role inthe pathogenesis of allergic rhinitis. So far, drug therapy is the focusof AR treatment, in which intranasal corticosteroids and antihistaminesare at the core position.

Atopic dermatitis (AD), also known as heterotopic dermatitis orhereditary allergic dermatitis, is a common dermatological disease thatis mostly seen in children and adolescents, and is often complicatedwith certain hereditary allergic diseases such as allergic rhinitis,asthma and the like. The involvement of immunological factors such asIL-4 and IL-13 are one of the main pathogenesis.

Eosinophilic esophagitis (EoE) is a chronic immune inflammatory diseasecharacterized by infiltration of eosinophils (EOS) in all layers ofesophagus. The onset of EoE is associated with dysfunction of Th2 cells.At present, protocols with high specificity, such as novel biologicalagent anti-IL-5 (such as mepolizumab) have become hotspots in research.Although immune-modulating therapy has achieved results in animalmodels, explorations are still needed in human clinical trials.Medicaments such as PGD2 inhibitors, anti-TNF-α, and anti-IL-13 areunder investigation at present.

Monoclonal antibody medicaments targeting the hIL-4R have now enteredclinical trials, such as Dupilumab, which has shown good efficacy inphase II clinical trial of atopic dermatitis. In addition to Dupilumab,other monoclonal antibodies against hIL-4R have been claimed in patentapplications by companies, for example U.S. Pat. Nos. 7,186,809 and7,638,606.

SUMMARY OF THE INVENTION

The present invention provides an antibody specific for IL-4R throughantibody screening and optimization, and the antibody can serve as ablocking agent for the binding of IL-4 to IL-4R, and can be used fortreating inflammation or allergic diseases by binding to IL-4R.

With reference to the numbering of amino acid residues of the sequenceas set forth in SEQ ID NO: 58, the antibody of the present invention hasa serine at position 31 (31Ser) of the light chain variable regioncomprised therein, and with reference to the numbering of amino acidresidues of the sequence as set forth in SEQ ID NO: 93, the antibody hasan aspartic acid at position 103 (103Asp), and a tyrosine at position104 (104Tyr) of the heavy chain variable region comprised therein.Wherein, the 31Ser is located in CDR1 of the light chain variable regionof the present antibody correspondingly, and the 103Asp and 104Tyr arelocated in the CDR3 of the heavy chain variable region of the presentantibody correspondingly.

Preferably, the light chain variable region of the present antibodycomprises CDR1 as set forth in SEQ ID NO: 2, and the heavy variableregion of the antibody comprises CDR3 as set forth in SEQ ID NO: 19.

Preferably, the light chain variable region of the present antibodycomprises a combination of CDR1, CDR2, and CDR3 selected from the groupconsisting of:

(1) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 3and CDR3 as set forth in SEQ ID NO: 5;

(2) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 4and CDR3 as set forth in SEQ ID NO: 5;

(3) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 3and CDR3 as set forth in SEQ ID NO: 6;

(4) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 3and CDR3 as set forth in SEQ ID NO: 7;

(5) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 3and CDR3 as set forth in SEQ ID NO: 8;

(6) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 4and CDR3 as set forth in SEQ ID NO: 6;

(7) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 4and CDR3 as set forth in SEQ ID NO: 7; and

(8) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 4and CDR3 as set forth in SEQ ID NO: 8; and

the heavy chain variable region of the present antibody comprises acombination of CDR1, CDR2, and CDR3 selected from the group consistingof:

(1) CDR1 as set forth in SEQ ID NO: 14, CDR2 as set forth in SEQ ID NO:17 and CDR3 as set forth in SEQ ID NO: 19;

(2) CDR1 as set forth in SEQ ID NO: 14, CDR2 as set forth in SEQ ID NO:18 and CDR3 as set forth in SEQ ID NO: 19;

(3) CDR1 as set forth in SEQ ID NO: 15, CDR2 as set forth in SEQ ID NO:17 and CDR3 as set forth in SEQ ID NO: 19;

(4) CDR1 as set forth in SEQ ID NO: 15, CDR2 as set forth in SEQ ID NO:

18 and CDR3 as set forth in SEQ ID NO: 19;

(5) CDR1 as set forth in SEQ ID NO: 16, CDR2 as set forth in SEQ ID NO:18 and CDR3 as set forth in SEQ ID NO: 19; and

(6) CDR1 as set forth in SEQ ID NO: 16, CDR2 as set forth in SEQ ID NO:17 and CDR3 as set forth in SEQ ID NO: 19.

More preferably, the present invention antibody comprises a light chainvariable region selected from the amino acid sequences shown in thefollowing sequences: SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 44, SEQ IDNO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQID NO: 51, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 57, and theantibody comprises a heavy chain variable region selected from the aminoacid sequences shown in the following sequences: SEQ ID NO: 59, SEQ IDNO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 74, SEQ ID NO: 75,SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO:80, SEQ ID NO: 91 and SEQ ID NO: 92.

Further preferably, the present antibody comprises a combination of alight chain variable region and a heavy chain variable region selectedfrom the group consisting of:

(1) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 62;

(2) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 63;

(3) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 59;

(4) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 60;

(5) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 61;

(6) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 67;

(7) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 65;

(8) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 66;

(9) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 64;

(10) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 91;

(11) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 74;

(12) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 75;

(13) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 76;

(14) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 77;

(15) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 78;

(16) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 80;

(17) a light chain variable region as set forth in SEQ ID NO: 40 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(18) a light chain variable region as set forth in SEQ ID NO: 41 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(19) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(20) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 62;

(21) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 91;

(22) a light chain variable region as set forth in SEQ ID NO: 49 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(23) a light chain variable region as set forth in SEQ ID NO: 50 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(24) a light chain variable region as set forth in SEQ ID NO: 45 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(25) a light chain variable region as set forth in SEQ ID NO: 46 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(26) a light chain variable region as set forth in SEQ ID NO: 47 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(27) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(28) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 62;

(29) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 91;

(30) a light chain variable region as set forth in SEQ ID NO: 54 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(31) a light chain variable region as set forth in SEQ ID NO: 54 and aheavy chain variable region as set forth in SEQ ID NO: 62;

(32) a light chain variable region as set forth in SEQ ID NO: 54 and aheavy chain variable region as set forth in SEQ ID NO: 91; and

(33) a light chain variable region as set forth in SEQ ID NO: 51 and aheavy chain variable region as set forth in SEQ ID NO: 92.

In another aspect, the present invention provides an antibody that canbind to Interleukin 4 (IL-4) receptor (IL-4R). The antibody comprises alight chain variable region (VL), and the light chain variable regioncomprises a combination of CDR1, CDR2 and CDR3 selected from the groupconsisting of:

(1) CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in SEQ ID NO: 3and CDR3 as set forth in SEQ ID NO: 5;

(2) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 3and CDR3 as set forth in SEQ ID NO: 5;

(3) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 4and CDR3 as set forth in SEQ ID NO: 5;

(4) CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in SEQ ID NO: 4and CDR3 as set forth in SEQ ID NO: 5;

(5) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 3and CDR3 as set forth in SEQ ID NO: 6;

(6) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 3and CDR3 as set forth in SEQ ID NO: 7;

(7) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 3and CDR3 as set forth in SEQ ID NO: 8;

(8) CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in SEQ ID NO: 3and CDR3 as set forth in SEQ ID NO: 6;

(9) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 4and CDR3 as set forth in SEQ ID NO: 6;

(10) CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO:4 and CDR3 as set forth in SEQ ID NO: 8;

(11) CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in SEQ ID NO:4 and CDR3 as set forth in SEQ ID NO: 8 and

(12) CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in SEQ ID NO:3 and CDR3 as set forth in SEQ ID NO: 8;

and/or

the antibody comprises a heavy chain variable region (VH), and the heavychain variable region comprises a combination of CDR1, CDR2 and CDR3selected from the group consisting of:

(1) CDR1 as set forth in SEQ ID NO: 14, CDR2 as set forth in SEQ ID NO:17 and CDR3 as set forth in SEQ ID NO: 19;

(2) CDR1 as set forth in SEQ ID NO: 14, CDR2 as set forth in SEQ ID NO:18 and CDR3 as set forth in SEQ ID NO: 19;

(3) CDR1 as set forth in SEQ ID NO: 14, CDR2 as set forth in SEQ ID NO:17 and CDR3 as set forth in SEQ ID NO: 20;

(4) CDR1 as set forth in SEQ ID NO: 14, CDR2 as set forth in SEQ ID NO:18 and CDR3 as set forth in SEQ ID NO: 20;

(5) CDR1 as set forth in SEQ ID NO: 15, CDR2 as set forth in SEQ ID NO:17 and CDR3 as set forth in SEQ ID NO: 19;

(6) CDR1 as set forth in SEQ ID NO: 16, CDR2 as set forth in SEQ ID NO:17 and CDR3 as set forth in SEQ ID NO: 19; and

(7) CDR1 as set forth in SEQ ID NO: 14, CDR2 as set forth in SEQ ID NO:18 and CDR3 as set forth in SEQ ID NO: 19.

Further, with regard to framework regions of the antibody of the presentinvention as described above, the light chain variable region of thepresent antibody preferably comprises a combination of FR1, FR2, FR3 andFR4 selected from the group consisting of:

(1) FR1 as set forth in SEQ ID NO: 9, FR2 as set forth in SEQ ID NO: 10,FR3 as set forth in SEQ ID NO: 12 and FR4 as set forth in SEQ ID NO: 13;and

(2) FR1 as set forth in SEQ ID NO: 9, FR2 as set forth in SEQ ID NO: 11,FR3 as set forth in SEQ ID NO: 12 and FR4 as set forth in SEQ ID NO: 13.

Preferably, the heavy chain variable region of the antibody comprises acombination of FR1, FR2, FR3 and FR4 selected from the group consistingof:

(1) FR1 as set forth in SEQ ID NO: 21, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(2) FR1 as set forth in SEQ ID NO: 22, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(3) FR1 as set forth in SEQ ID NO: 23, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(4) FR1 as set forth in SEQ ID NO: 24, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(5) FR1 as set forth in SEQ ID NO: 24, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 35 and FR4 as set forth in SEQ ID NO:38;

(6) FR1 as set forth in SEQ ID NO: 25, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(7) FR1 as set forth in SEQ ID NO: 26, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(8) FR1 as set forth in SEQ ID NO: 27, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(9) FR1 as set forth in SEQ ID NO: 29, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(10) FR1 as set forth in SEQ ID NO: 30, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(11) FR1 as set forth in SEQ ID NO: 24, FR2 as set forth in SEQ ID NO:33, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(12) FR1 as set forth in SEQ ID NO: 24, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 36 and FR4 as set forth in SEQ ID NO:38;

(13) FR1 as set forth in SEQ ID NO: 24, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 37 and FR4 as set forth in SEQ ID NO:38;

(14) FR1 as set forth in SEQ ID NO: 31, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(15) FR1 as set forth in SEQ ID NO: 27, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 35 and FR4 as set forth in SEQ ID NO:38;

(16) FR1 as set forth in SEQ ID NO: 26, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 35 and FR4 as set forth in SEQ ID NO:38;

(17) FR1 as set forth in SEQ ID NO: 25, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 35 and FR4 as set forth in SEQ ID NO:38;

(18) FR1 as set forth in SEQ ID NO: 28, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 35 and FR4 as set forth in SEQ ID NO:38;

(19) FR1 as set forth in SEQ ID NO: 28, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 34 and FR4 as set forth in SEQ ID NO:38;

(20) FR1 as set forth in SEQ ID NO: 23, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 35 and FR4 as set forth in SEQ ID NO:38;

(21) FR1 as set forth in SEQ ID NO: 22, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 35 and FR4 as set forth in SEQ ID NO:38; and

(22) FR1 as set forth in SEQ ID NO: 21, FR2 as set forth in SEQ ID NO:32, FR3 as set forth in SEQ ID NO: 35 and FR4 as set forth in SEQ ID NO:38.

According to the domain composition of a light chain variable region anda heavy chain variable region of an antibody known in the art, the lightchain variable region or the heavy chain variable region of presentantibody comprises the above domain components in an order ofFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, or comprises the above domain componentsin an order of(X)n-FR1-(X)n-CDR1-(X)n-FR2-(X)n-CDR2-(X)n-FR3-(X)n-CDR3-(X)n-FR4-(X)n,in which, X is any amino acid residue, and n is zero or an integergreater than zero.

Preferably, the antibody provided by the present invention comprises alight chain variable region selected from the amino acid sequences shownin the following sequences:

SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57and SEQ ID NO: 58;

and/or

the antibody provided by the present invention comprises a heavy chainvariable region selected from the amino acid sequences shown in thefollowing sequences:

SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO:63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ IDNO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77,SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ IDNO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQID NO: 92 and SEQ ID NO: 93.

According to particular embodiments of the present invention, theantibody provided by the present invention comprises a combination of alight chain variable region and a heavy chain variable region selectedfrom the group consisting of:

(1) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 62;

(2) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 63;

(3) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 59;

(4) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 60;

(5) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 61;

(6) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 67;

(7) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 65;

(8) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 66;

(9) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 64;

(10) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 68;

(11) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 69;

(12) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 70;

(13) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 71;

(14) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 72;

(15) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 73;

(16) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 89;

(17) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 88;

(18) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 87;

(19) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 86;

(20) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 83;

(21) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 82;

(22) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 81;

(23) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 85;

(24) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 84;

(25) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 91;

(26) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 90;

(27) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 74;

(28) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 75;

(29) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 76;

(30) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 77;

(31) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 78;

(32) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 80;

(33) a light chain variable region as set forth in SEQ ID NO: 39 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(34) a light chain variable region as set forth in SEQ ID NO: 40 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(35) a light chain variable region as set forth in SEQ ID NO: 41 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(36) a light chain variable region as set forth in SEQ ID NO: 42 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(37) a light chain variable region as set forth in SEQ ID NO: 43 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(38) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(39) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 62;

(40) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 68;

(41) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 72;

(42) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 82;

(43) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 85;

(44) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 91;

(45) a light chain variable region as set forth in SEQ ID NO: 48 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(46) a light chain variable region as set forth in SEQ ID NO: 49 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(47) a light chain variable region as set forth in SEQ ID NO: 50 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(48) a light chain variable region as set forth in SEQ ID NO: 45 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(49) a light chain variable region as set forth in SEQ ID NO: 46 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(50) a light chain variable region as set forth in SEQ ID NO: 47 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(51) a light chain variable region as set forth in SEQ ID NO: 56 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(52) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(53) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 62;

(54) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 68;

(55) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 72;

(56) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 82;

(57) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 85;

(58) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 91;

(59) a light chain variable region as set forth in SEQ ID NO: 54 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(60) a light chain variable region as set forth in SEQ ID NO: 54 and aheavy chain variable region as set forth in SEQ ID NO: 62;

(61) a light chain variable region as set forth in SEQ ID NO: 54 and aheavy chain variable region as set forth in SEQ ID NO: 68;

(62) a light chain variable region as set forth in SEQ ID NO: 54 and aheavy chain variable region as set forth in SEQ ID NO: 72;

(63) a light chain variable region as set forth in SEQ ID NO: 54 and aheavy chain variable region as set forth in SEQ ID NO: 82;

(64) a light chain variable region as set forth in SEQ ID NO: 54 and aheavy chain variable region as set forth in SEQ ID NO: 85;

(65) a light chain variable region as set forth in SEQ ID NO: 54 and aheavy chain variable region as set forth in SEQ ID NO: 91;

(66) a light chain variable region as set forth in SEQ ID NO: 53 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(67) a light chain variable region as set forth in SEQ ID NO: 51 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(68) a light chain variable region as set forth in SEQ ID NO: 52 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(69) a light chain variable region as set forth in SEQ ID NO: 52 and aheavy chain variable region as set forth in SEQ ID NO: 62;

(70) a light chain variable region as set forth in SEQ ID NO: 52 and aheavy chain variable region as set forth in SEQ ID NO: 91;

(71) a light chain variable region as set forth in SEQ ID NO: 58 and aheavy chain variable region as set forth in SEQ ID NO: 92;

(72) a light chain variable region as set forth in SEQ ID NO: 57 and aheavy chain variable region as set forth in SEQ ID NO: 93;

(73) a light chain variable region as set forth in SEQ ID NO: 44 and aheavy chain variable region as set forth in SEQ ID NO: 93;

(74) a light chain variable region as set forth in SEQ ID NO: 58 and aheavy chain variable region as set forth in SEQ ID NO: 91;

(75) a light chain variable region as set forth in SEQ ID NO: 55 and aheavy chain variable region as set forth in SEQ ID NO: 93; and

(76) a light chain variable region as set forth in SEQ ID NO: 51 and aheavy chain variable region as set forth in SEQ ID NO: 93.

The antibody provided by the present invention is capable of binding toIL-4R and functions as an antagonist of IL-4R. Preferably, the antibodyis capable of binding to IL-4Rα, preferably to mammal IL-4Rα, morepreferably to human IL-4Rα, even more preferably to human solubleIL-4Rα.

Binding affinity of the antibody provided by the present invention toIL-4Rα can be determined by method Biacore or ELISA. The antibody isdetermined to bind IL-4Rα with an affinity of less than 100 nM, lessthan 10 nM, less than 1 nM, less than 0.5 nM, and even less than 0.1 nM.

Under the same conditions, the ratio of the expression level of theantibody provided by the present invention to that of reference antibodyis 0.1-3:1, preferably 0.3-3:1, more preferably 0.4-3:1, stillpreferably 0.5-3:1, still more preferably 0.6-3:1, further preferably0.7-3:1, further more preferably 1-3:1.

In terms of antibody type, the antibody provided by the presentinvention can be a monoclonal antibody, fully or partially humanizedantibody or chimeric antibody.

Or preferably, the antibody is an immunoglobulin, preferably IgA, IgD,IgE, IgG or IgM, more preferably IgG1, IgG2, IgG3 or IgG4 subtype,further more preferably IgG2 or IgG4 subtype.

In further another aspect, the invention provides a fusion protein orconjugate comprising the antibody of the present invention. The fusionprotein or conjugate may further comprises cell surface receptor, activeprotein or active polypeptide, small molecule compound such as aminoacid and saccharide, small molecule polymer or other parts chemicallymodifying the antibody, etc., bound to the present antibody by chemicalor physical means.

In still another aspect, the present invention provides a nucleic acidsequence that can encode the heavy chain variable region and/or lightchain variable region of the antibody provided by the present invention.

Preferably, the nucleic acid sequence can encode the heavy chain and/orlight chain of the antibody provided by the present invention.

In yet another aspect, the present invention also provides a vectorcomprising the nucleic acid sequence provided by the present invention.The vector may be an eukaryotic expression vector, a prokaryoticexpression vector, an artificial chromosome, a phage vector or the like.

The vector or nucleic acid sequence as described above can be used totransform or transfect host cells for purposes of preservation orantibody expression, and the like. Accordingly, the invention alsoprovides a host cell transformed or transfected with the nucleic acidsequence or the vector. The host cell can be any prokaryotic oreukaryotic cell, such as a bacterial or insect, fungal, plant or animalcell.

The antibody provided by the present invention can be obtained throughany method known in the art. For example, the heavy chain variableregion and/or the light chain variable region of the antibody, or theheavy and/or light chain of the antibody can be obtained from thenucleic acid sequence provided by the present invention firstly, andthen assembled into the antibody with any other domain of the antibody;or, the host cell provided by the present invention is cultured underconditions that the host cell is allowed to express the heavy chainvariable region and/or the light chain variable region of the antibodyor the heavy and/or light chain of the antibody for assembly into theantibody.

Optionally, the method further includes a step of recovering theproduced antibody.

The antibody, fusion protein or conjugate, nucleic acid sequence, vectoror host cell provided by the present invention or antibody produced bythe method as described above may be contained in a pharmaceuticalcomposition, and more particularly, in a pharmaceutical preparation forvarious purposes as actually needed. Therefore, in a further aspect, thepresent invention also provides a pharmaceutical composition comprisingthe antibody, fusion protein or conjugate, nucleic acid sequence,vector, host cell of the present invention and/or antibody produced bythe method as described above.

Optionally, the pharmaceutical composition can be a pharmaceuticalpreparation. The pharmaceutical preparation is, for example, aninjection.

The pharmaceutical composition or pharmaceutical preparation may furthercomprises a pharmaceutically acceptable carrier or excipient, dependingon the particular dosage form.

In the pharmaceutical composition or pharmaceutical preparation, atleast one of the following medicaments may also be contained:antiasthmatics such as albuterol etc., antihistamines such as loratadineetc., Immunosuppressive agents such as tacrolimus and pimecrolimus etc.,M receptor blockers such as ipratropium bromide etc., leukotrienereceptor blockers such as montelukast etc., phosphodiesterase inhibitorssuch as theophylline etc., non-steroidal anti-inflammatory drugs such as5-aminosalicylic acid etc., hormones such as beclomethasone andbudesonide etc., that is to say the antibody, fusion protein orconjugate, nucleic acid sequence, vector, host cell provided by thepreset invention or antibody produced by the present method as describedabove may be used in combination with other medicaments as required.

In still further another aspect, the present invention also provides useof the antibody, fusion protein or conjugate, nucleic acid sequence,vector, host cell or antibody produced by the method for manufacturing amedicament for the prevention, treatment or amelioration of inflammationor allergic disease.

Preferably, the inflammation or allergic disease includes autoimmunedisease, such as allergic dermatitis, asthma, eosinophilic esophagitis,eczema, allergic rhinitis, nasal polyp, rheumatoid arthritis and thelike.

In still yet another aspect, the present invention provides a method ofpreventing, treating or ameliorating inflammation or allergic disease,and the method includes administering to a subject in need thereof theantibody, fusion protein or conjugate, nucleic acid sequence, vector,host cell provided by the present invention and/or antibody produced bythe present method.

Preferably, the subject is a mammal, more preferably, the subject is ahuman.

Preferably, the inflammation or allergic disease includes autoimmunedisease, such as allergic dermatitis, asthma, eosinophilic esophagitis,eczema, allergic rhinitis, nasal polyp, rheumatoid arthritis and thelike.

Other medicaments can be used in combination to prevent, treat orameliorate inflammation or allergic disease, for example, the methodfurther includes administering to a subject in need thereof at least onemedicament selected from the group consisting of: antiasthmatics such asalbuterol etc., antihistamines such as loratadine etc.,Immunosuppressive agents such as tacrolimus and pimecrolimus etc., Mreceptor blockers such as ipratropium bromide etc., leukotriene receptorblockers such as montelukast etc., phosphodiesterase inhibitors such astheophylline etc., non-steroidal anti-inflammatory drugs such as5-aminosalicylic acid, etc., hormones such as beclomethasone andbudesonide etc.

Preferably, the medicament is administered simultaneously orsequentially with the antibody, fusion protein or conjugate, nucleicacid sequence, vector, host cell provided by the present inventionand/or antibody produced by the present method.

In further yet another aspect, the present invention also provides akit, and the kit comprises the antibody, fusion protein or conjugate,nucleic acid sequence, vector, host cell provided by the presentinvention and/or antibody produced by the present method.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be better understood by combination with thenon-limiting embodiments of the present invention and the accompanyingdrawings, in which:

FIG. 1 shows the pharmacokinetic curves of the antibodies of the presentinvention in mouse.

FIG. 2 shows the pharmacokinetic curves of the antibodies of the presentinvention in Macaca fascicularis.

FIG. 3 includes panels 3A-3C which show the specific binding of theantibodies of the present invention with TF-1 cells expressing IL-4Rα,as determined by FACS, in which panel 3A shows the fluorescence signalwithout the addition of antibody L1012H1031 of the present invention;panel 3B shows the fluorescence signal with the addition of antibodyL1012H1031 of the present invention; and panel 3C shows the signalsuperposition of panels 3A and 3B for comparison.

FIG. 4 includes panels 4A-4D which show that the specific binding of theantibodies of the present invention with TF-1 cells expressing IL-4Rα isblocked by sIL-4Rα present in the system, as determined by FACS, inwhich panel 4A shows the fluorescence signal without the addition ofantibody L1012H1031 of the present invention; panel 4B shows thefluorescence signal with the addition of antibody L1012H1031 of thepresent invention; panel 4C shows the fluorescence signal with theaddition of antibody L1012H1031 of the present invention and sIL-4Rα,and panel 4D shows the signal superposition of panels 4A-4C forcomparison.

FIG. 5 includes panels 5A-5B which show the effect of the antibodies ofthe present invention on the inhibition of TARO and MDC release, asdetermined by ELISA, in which panel 5A shows the result that antibodyL1020H1031 of the present invention inhibits TARO release, and panel 5Bshows the result that antibody L1020H1031 of the present inventioninhibits MDC release.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention will be further described in detail in combinationwith the particular embodiments hereinafter. It will be appreciated bythose skilled in the art that the embodiments provided are only used toillustrate the present invention, rather than limiting the scope of thepresent invention in any way.

Experimental methods in the following Examples are all conventionalmethods, unless particularly stated. Raw materials used in the followingExamples are commercially available from conventional biochemicalreagent stores, unless particularly stated.

In the following Examples, the antibodies shown in Table 1 and theireffects are exemplarily provided and verified.

TABLE 1 Exemplary antibodies provided by the present invention SED IDNOs of the light chain variable Number region and the heavy chainvariable region L1000H1007 SEQ ID NO:: 57 + SEQ ID NO:: 62 L1000H1008SEQ ID NO:: 57 + SEQ ID NO:: 63 L1000H1009 SEQ ID NO:: 57 + SEQ ID NO:59 L1000H1010 SEQ ID NO: 57 + SEQ ID NO: 60 L1000H1011 SEQ ID NO: 57 +SEQ ID NO: 61 L1000H1012 SEQ ID NO: 57 + SEQ ID NO: 67 L1000H1013 SEQ IDNO: 57 + SEQ ID NO: 65 L1000H1014 SEQ ID NO: 57 + SEQ ID NO: 66L1000H1015 SEQ ID NO: 57 + SEQ ID NO: 64 L1000H1016 SEQ ID NO: 57 + SEQID NO: 68 L1000H1017 SEQ ID NO: 57 + SEQ ID NO: 69 L1000H1018 SEQ ID NO:57 + SEQ ID NO: 70 L1000H1019 SEQ ID NO: 57 + SEQ ID NO: 71 L1000H1020SEQ ID NO: 57 + SEQ ID NO: 72 L1000H1021 SEQ ID NO: 57 + SEQ ID NO: 73L1000H1022 SEQ ID NO: 57 + SEQ ID NO: 89 L1000H1023 SEQ ID NO: 57 + SEQID NO: 88 L1000H1024 SEQ ID NO: 57 + SEQ ID NO: 87 L1000H1025 SEQ ID NO:57 + SEQ ID NO: 86 L1000H1026 SEQ ID NO: 57 + SEQ ID NO: 83 L1000H1027SEQ ID NO: 57 + SEQ ID NO: 82 L1000H1028 SEQ ID NO: 57 + SEQ ID NO: 81L1000H1029 SEQ ID NO: 57 + SEQ ID NO: 85 L1000H1030 SEQ ID NO: 57 + SEQID NO: 84 L1000H1031 SEQ ID NO: 57 + SEQ ID NO: 91 L1000H1032 SEQ ID NO:57 + SEQ ID NO: 90 L1000H1033 SEQ ID NO: 57 + SEQ ID NO: 74 L1000H1034SEQ ID NO: 57 + SEQ ID NO: 75 L1000H1035 SEQ ID NO: 57 + SEQ ID NO: 76L1000H1036 SEQ ID NO: 57 + SEQ ID NO: 77 L1000H1037 SEQ ID NO: 57 + SEQID NO: 78 L1000H1038 SEQ ID NO: 57 + SEQ ID NO: 80 L1007H1000 SEQ ID NO:39 + SEQ ID NO: 92 L1008H1000 SEQ ID NO: 40 + SEQ ID NO: 92 L1009H1000SEQ ID NO: 41 + SEQ ID NO: 92 L1010H1000 SEQ ID NO: 42 + SEQ ID NO: 92L1011H1000 SEQ ID NO: 43 + SEQ ID NO: 92 L1012H1000 SEQ ID NO: 44 + SEQID NO: 92 L1012H1007 SEQ ID NO: 44 + SEQ ID NO: 62 L1012H1016 SEQ ID NO:44 + SEQ ID NO: 68 L1012H1020 SEQ ID NO: 44 + SEQ ID NO: 72 L1012H1027SEQ ID NO: 44 + SEQ ID NO: 82 L1012H1029 SEQ ID NO: 44 + SEQ ID NO: 85L1012H1031 SEQ ID NO: 44 + SEQ ID NO: 91 L1013H1000 SEQ ID NO: 48 + SEQID NO: 92 L1014H1000 SEQ ID NO: 49 + SEQ ID NO: 92 L1015H1000 SEQ ID NO:50 + SEQ ID NO: 92 L1016H1000 SEQ ID NO: 45 + SEQ ID NO: 92 L1017H1000SEQ ID NO: 46 + SEQ ID NO: 92 L1018H1000 SEQ ID NO: 47 + SEQ ID NO: 92L1019H1000 SEQ ID NO: 56 + SEQ ID NO: 92 L1020H1000 SEQ ID NO: 55 + SEQID NO: 92 L1020H1007 SEQ ID NO: 55 + SEQ ID NO: 62 L1020H1016 SEQ ID NO:55 + SEQ ID NO: 68 L1020H1020 SEQ ID NO: 55 + SEQ ID NO: 72 L1020H1027SEQ ID NO: 55 + SEQ ID NO: 82 L1020H1029 SEQ ID NO: 55 + SEQ ID NO: 85L1020H1031 SEQ ID NO: 55 + SEQ ID NO: 91 L1021H1000 SEQ ID NO: 54 + SEQID NO: 92 L1021H1007 SEQ ID NO: 54 + SEQ ID NO: 62 L1021H1016 SEQ ID NO:54 + SEQ ID NO: 68 L1021H1020 SEQ ID NO: 54 + SEQ ID NO: 72 L1021H1027SEQ ID NO: 54 + SEQ ID NO: 82 L1021H1029 SEQ ID NO: 54 + SEQ ID NO: 85L1021H1031 SEQ ID NO: 54 + SEQ ID NO: 91 L1022H1000 SEQ ID NO: 53 + SEQID NO: 92 L1023H1000 SEQ ID NO: 51 + SEQ ID NO: 92 L1024H1000 SEQ ID NO:52 + SEQ ID NO: 92 L1024H1007 SEQ ID NO: 52 + SEQ ID NO: 62 L1024H1031SEQ ID NO: 52 + SEQ ID NO: 91 L1001H1000 SEQ ID NO: 58 + SEQ ID NO: 92L1000H1001 SEQ ID NO: 57 + SEQ ID NO: 93 L1012H1001 SEQ ID NO: 44 + SEQID NO: 93 L1001H1031 SEQ ID NO: 58 + SEQ ID NO: 91 L1020H1001 SEQ ID NO:55 + SEQ ID NO: 93 L1023H1001 SEQ ID NO: 51 + SEQ ID NO: 93 L1000H1000SEQ ID NO: 57 + SEQ ID NO: 92 L1001H1001 SEQ ID NO: 58 + SEQ ID NO: 93

Example 1: Preparation of the Antibodies of the Present Invention andDetermination of Expression Levels by Non-Reduced SDS-PAGE GelElectrophoresis

The sequence coding for the light chain variable region of the antibodywas inserted into vector pFUSE2ss-CLIg-hK (Invivogen, Catalog Number:pfuse2ss-hclk) using EcoRI and BsiWI restriction sites to construct alight chain expression vector. The sequence coding for the heavy chainvariable region of the antibody was inserted into vectorpFUSEss-CHIg-hG2 (Invivogen, Catalog Number: pfusess-hchg2) or vectorpFUSEss-CHIg-hG4 (Invivogen, Catalog Number: pfusess-hchg4) using EcoRIand NheI restriction sites to construct a heavy chain expression vector.

The culture and transfection of Expi293 cells were performed inaccordance with the handbook of Expi293™ Expression System Kit fromInvitrogen (Catalog Number: A14635). The density of the cells wasadjusted to 2×10⁶ cells/ml for transfection, and 0.6 μg of the lightchain expression vector as described above and 0.4 μg of the heavy chainexpression vector as described above were added to each ml of cellculture, and the supernatant of the culture was collected four dayslater.

The culture supernatant was subjected to non-reduced SDS-PAGE gelelectrophoresis in accordance with the protocol described in Appendix 8,the Third edition of the “Molecular Cloning: A Laboratory Manual”.

Pictures were taken with a gel scanning imaging system from BEIJINGJUNYI Electrophoresis Co., LTD and in-gel quantification was performedusing Gel-PRO ANALYZER software to determine the expression levels ofthe antibodies after transient transfection. Results were expressedrelative to the expression level of control antibody 1 (control antibody1 was constructed according to U.S. Pat. No. 7,186,809, which comprisesa light chain variable region as set forth in SEQ ID NO: 10 of U.S. Pat.No. 7,186,809 and a heavy chain variable region as set forth in SEQ IDNO: 12 of U.S. Pat. No. 7,186,809, the same below) (control antibody 2was constructed according to U.S. Pat. No. 7,638,606, which comprises alight chain variable region as set forth in SEQ ID NO: 6 of U.S. Pat.No. 7,638,606 and a variable region as set forth in SEQ ID NO: 42 ofU.S. Pat. No. 7,638,606, the same below). See Tables 2a-2c below for theresults.

TABLE 2a Expression levels of the antibodies of the present inventionafter transient transfection (antibodies whose expression levels aresignificantly higher than that of control antibody 1): Number ofExpression level vs Number of Expression level vs the antibody controlantibody 1 the antibody control antibody 1 L1021H1000 2.08 L1000H10281.27 L1020H1000 1.58 L1000H1015 1.19 L1000H1027 1.56 L1000H1032 1.18L1000H1024 1.51 L1000H1026 1.15 L1000H1025 1.48 L1021H1029 1.12L1001H1000 1.48 L1000H1030 1.1 L1021H1016 1.43 L1024H1031 1.08L1000H1014 1.35 L1000H1016 1.05

TABLE 2b Expression levels of the antibodies of the present inventionafter transient transfection (antibodies whose expression levels areslightly lower than that of control antibody 1): Number of Expressionlevel vs Number of Expression level vs the antibody control antibody 1the antibody control antibody 1 L1000H1031 0.99 L1017H1000 0.85L1021H1031 0.99 L1020H1016 0.84 L1020H1029 0.96 L1000H1009 0.81 controlanti- 0.93 L1000H1007 0.8 body 2 L1012H1000 0.89 L1000H1023 0.8L1019H1000 0.87 L1020H1027 0.78 L1020H1031 0.87 L1024H1007 0.77L1021H1020 0.87 L1000H1013 0.75 L1000H1029 0.86 L1020H1007 0.74L1008H1000 0.86 L1021H1007 0.74 L1000H1001 0.85 L1000H1021 0.71

TABLE 2c Expression levels of the antibodies of the present inventionafter transient transfection (antibodies whose expression levels aresignificantly lower than that of control antibody 1): Number ofExpression level vs Number of Expression level vs the antibody controlantibody 1 the antibody control antibody 1 L1000H1020 0.69 L1024H10000.52 L1010H1000 0.69 L1000H1008 0.51 L1000H1022 0.67 L1000H1037 0.5L1000H1012 0.64 L1007H1000 0.49 L1022H1000 0.64 L1016H1000 0.49L1011H1000 0.63 L1000H1017 0.47 L1000H1011 0.62 L1000H1035 0.46L1000H1033 0.62 L1012H1027 0.46 L1020H1020 0.61 L1018H1000 0.44L1000H1036 0.6 L1023H1000 0.43 L1021H1027 0.6 L1012H1016 0.42 L1012H10070.59 L1013H1000 0.41 L1009H1000 0.57 L1000H1034 0.4 L1012H1020 0.57L1000H1018 0.35 L1012H1031 0.56 L1000H1019 0.34 L1000H1038 0.54L1015H1000 0.27 L1012H1029 0.54 L1014H1000 0.17 L1000H1010 0.53

Example 2: Detection of Inhibitory Effect of the Antibodies of thePresent Invention on Proliferation of TF-1 Cells by hIL-4 or hIL-13

1. Preparation of Reagents

hIL-4 (Invivogen, Catalog Number: rhil-4) solution: hIL-4 was dissolvedin 100 μl of PBS containing 0.1% BSA (Beyotime, Catalog Number: ST023)to obtain a solution with a concentration of 100 μg/ml, and thedissolved hIL-4 was dispensed into 1.5 ml (Nunc) centrifuge tubes in avolume of 5 μl per tube, and then the tubes were stored in arefrigerator at −20° C.

WST-1 (Beyotime, Catalog Number: C0036) solution: 5 ml of electroncoupling agent (C0036-2) was added to WST-1 powder (C0036-1), and theWST-1 solution was obtained when the WST-1 powder was dissolvedcompletely, then dispensed into 1.5 ml (Nunc) centrifuge tubes in avolume of 620 μl per tube, and then the tubes were stored in arefrigerator at −20° C.

2. Culture of TF-1 Cells

TF-1 cells (ATCC: CRL-2003™) frozen in liquid nitrogen were taken out,shaken in a 37° C. water bath to dissolve quickly. The dissolved cellsuspension was transferred to a 15 ml centrifuge tube, and then 1640medium was added therein to 10 ml. The tube was centrifuged at 800 rpmfor 5 min, the supernatant therein was aspirated, and cell pellets wereretained and washed once again. 10 ml of 1640 medium containing 10% FBSand 2 ng/ml GM-CSF (Sino Biological, Catalog Number: 10015-H01H) wasadded to the tube, and a cell density of 1×10⁵-1×10⁶ cells/ml wasobtained. The suspension was transferred to a T75 cell culture flask(Nunc), and the cells were statically cultured in an incubator (Thermo)at 37° C., 5% CO₂. Every 2-3 days, the cell suspension was taken out,centrifuged at 800 rpm for 5 min, and the cells were resuspended in 10ml of medium. Subsequently, 1×10⁶ cells were counted and transferredinto a new T75 cell culture flask, and the medium was supplementedthereto to 10 ml. The cells were continuously passaged for 2-3 times toreach a good state (the cells were bright and had a slightly irregularshape when suspended individually) for proliferation blockingexperiment.

3. Preparation and Purification of the Antibodies

a) Cell Culture Supernatant Samples of the Antibodies of the PresentInvention:

Expi293 cells were transfected with plasmids carrying different groupsof genes of the antibodies, and 200 μl of the cell culture supernatantwas taken out 4 days after transfection, and then centrifuged at 800 rpmfor 5 min. The supernatant was filtered through a 0.22 μm pore sizefilter and used for proliferation blocking experiment.

b) Purified antibody samples of the present invention: For each antibodyof the present invention, the culture supernatant of cells expressingthe antibody was passed through a 0.22 μm filter and then purified by GEMabSelect Sure (Catalog Number: 11003494) Protein A affinitychromatography column in a purification system GE AKTA purifier 10. Thepurified antibody was collected and concentrated using Amiconultrafiltration concentrator (Catalog Number: UFC903096) and thenquantified. The antibody was diluted with PBS to 0 to 1 μg/ml for theproliferation blocking experiment.

4. Proliferation Blocking Experiment

The well-grown cells in the T75 cell flask were taken out andtransferred into a 15 ml centrifuge tube, which was then centrifuged at800 rpm for 5 min. The supernatant was discarded and the cell pelletswere resuspended with 10 ml PBS, centrifuged at 800 rpm for 5 min. Thesupernatant was discarded and the cell pellets were resuspended in 10 mlof 1640 medium (without GM-CSF) containing 10% FBS, centrifuged at 800rpm for 5 min. The supernatant was discarded and the cell pellets wereresuspended in 5 ml of 1640 medium (without GM-CSF) containing 10% FBS.The cells were counted and adjusted to a cell density of 5×10⁵ cells/mlthrough supplementing the medium. The cell suspension was added to a96-well plate at a volume of 80 μl per well (wells in the outer ringwere left free of cells to prevent volatilization). For each antibody ofthe present invention, 10 μl of purified antibody with differentconcentrations or 10 μl of the corresponding cell culture supernatantwas added to the cells in the 96-well plate (in 3 replicate wells). ThehIL-4 was then diluted to 50 ng/ml with 1640 medium containing 10% FBS,and was added to the corresponding wells in the 96-well plate in avolume of 10 μl per well, so that the final cell density was 4×10⁵cells/mi, the concentration of hIL-4 was 5 ng/ml, and the volume in eachwell of the 96-well plate was 100 μl. A negative control group (in 3replicate wells) was set, in which no hIL-4 or the antibody was added,and only the same number of cells and the same volume of the culturemedium were added. Meanwhile, a positive control group (in 3 replicatewells) with addition of the same concentration of hIL-4 and the samevolume of the medium was set, and no antibody was added in in thisgroup. 200 μl of PBS was added to each well in the outer ring of the96-well cell plate to prevent the volatilization of liquid in the innerring. The 96-well cell plate was placed in a 5% CO₂ incubator at 37° C.for static culture.

The above experiment was repeated with the same procedure using 500ng/ml of hIL-13 (its final concentration was 50 ng/ml after being addedinto the cells).

5. Data Statistics

After the 96-well cell plates were statically cultured for 72 hours inthe 5% CO₂ incubator, 10 μl of the WST-1 solution was added to the cellsin each well. The 96-well cell plate was placed in a 5% CO2 incubator at37° C. for further static culture. 24 h later, the 96-well plate wasplaced in flexstation 3 (Molecular Devices) and the values ofOD450-OD650 were read.

For the culture supernatants of the antibodies of the invention, thevalues of OD450-OD650 (OD value) of them and those of the positivecontrol group and the negative control group were used to calculateinhibitory rate as follows: inhibitory rate=(OD value of supernatant ofthe transfected cells−OD value of the positive control group)/(OD valueof the negative control group−OD value of the positive controlgroup)×100%. The results of blocking effect of the antibodies of thepresent invention on the proliferation of TF-1 cells by hIL-4 or hIL-13are shown in Tables 3a-3b below.

For the purified antibodies of the present invention with differentconcentrations, the measured OD450-OD650 data were input into prism5software, in which the value of the negative control group was set tothe lowest one, the value of the positive control group was set to thehighest one, and the logarithm of the antibody concentration was taken.Then the curve of the antibody concentration logarithm to theOD450-OD650 value was fitted by prism5 software. The calculated IC50sare shown in Table 4 below.

TABLE 3a Screening results of inhibitory effects of the antibodies ofthe present invention on the proliferation activity of TF-1 cells(antibodies with increased inhibitory rates compared with controlantibody 1). Number Inhibitory rate on Inhibitory rate on of thepro-proliferative effect pro-proliferative effect antibody of IL-4 ofIL-13 control antibody 1 0.87 0.63 control antibody 2 1 0.87 L1020H10291.03 0.93 L1000H1001 1.03 0.93 L1021H1027 1.02 0.94 L1020H1027 1.02 0.92L1021H1029 1.02 0.92 L1020H1016 1.01 0.97 L1024H1031 1.01 0.94L1021H1031 1.01 0.93 L1020H1031 1.01 0.93 L1000H1029 1.01 0.92L1000H1027 1.01 0.86 L1021H1016 1 0.94 L1021H1007 1 0.86 L1000H1028 10.84 L1000H1014 0.99 0.78 L1001H1000 0.98 0.85 L1000H1024 0.98 0.75L1000H1031 0.97 0.83 L1000H1007 0.96 0.82 L1000H1023 0.96 0.8 L1000H10320.96 0.74 L1020H1007 0.95 0.9 L1021H1000 0.95 0.78 L1000H1016 0.95 0.76L1012H1029 0.94 0.95 L1012H1007 0.93 0.88 L1021H1020 0.91 0.75L1020H1000 0.91 0.7 L1000H1025 0.91 0.69 L1012H1020 0.9 0.78 L1000H10200.88 0.76 L1000H1012 0.88 0.58 L1020H1020 0.87 0.76

TABLE 3b Screening results of inhibitory effects of the antibodies ofthe present invention on the proliferation activity of TF-1 cells(antibodies with the same or decreased inhibitory rates compared withcontrol antibody 1). Number Inhibitory rate on Inhibitory rate on of thepro-proliferative effect pro-proliferative effect antibody of IL-4 ofIL-13 L1000H1022 0.87 0.62 L1000H1017 0.87 0.59 L1000H1030 0.87 0.47L1024H1007 0.86 0.7 L1000H1013 0.86 0.55 L1012H1031 0.85 0.7 L1000H10150.85 0.63 L1000H1026 0.85 0.53 L1000H1018 0.84 0.65 L1000H1009 0.83 0.54L1012H1000 0.78 0.5 L1012H1016 0.78 0.48 L1000H1021 0.73 0.56 L1019H10000.72 0.49 L1008H1000 0.67 0.27 L1009H1000 0.66 0.35 L1000H1008 0.66 0.19L1012H1027 0.65 0.63 L1000H1011 0.61 0.26 L1000H1010 0.54 0.1 L1000H10190.52 0.29 L1007H1000 0.49 0.15 L1024H1000 0.44 0.39 L1011H1000 0.44 0.13L1017H1000 0.44 0.02 L1018H1000 0.42 −0.12 L1010H1000 0.38 0.06L1000H1036 0.27 0.18 L1023H1000 0.26 0.07 L1000H1033 0.23 0.14L1015H1000 0.16 0.09 L1000H1034 0.15 0.1 L1016H1000 0.1 −0.36 L1013H10000.01 −0.21 L1022H1000 −0.05 0.02 L1000H1038 −0.05 −0.15 L1014H1000 −0.05−0.29 L1000H1037 −0.06 −0.13 L1000H1035 −0.12 −0.13

TABLE 4 Activity data of the purified antibodies (600-900 ml) aftertransient transfection IC50 (ng/ml) IC50 (ng/ml) (Inhibition on the(Inhibition on the proliferation of IL-4) proliferation of IL-13)control antibody 1 184.03 ± 61.95  345.1 ± 73.9  control antibody 2 27.79 ± 3.22   52.06 ± 14.97 L1021H1016  28.46 ± 7.82   69.67 ± 28.38L1021H1020  15.36 ± 2.46   28.64 ± 10.46 L1021H1031  34.27 ± 12.17 76.51 ± 31.94 L1020H1016  24.19 ± 6.91   44.62 ± 14.79 L1020H1020  23.63± 8.73   53.73 ± 17.34 L1020H1031   25.6 ± 7.46   60.17 ± 19.02L1012H1016  32.44 ± 7.45   75.33 ± 43.63 L1012H1020  21.09 ± 4.72  56.65 ± 23.34 L1012H1031  39.26 ± 15.61  76.87 ± 40.97

Example 3 Detection of the Binding Abilities of the Antibodies of thePresent Invention to sIL-4Rα by ELISA

1. Preparation of Reagents

sIL-4Rα (PEPRO TECH, Catalog Number: 200-04R) solution: the sIL-4Rα wastaken and 1 ml ddH2O was added therein, mixed up and down, and then asolution of 100 μg/ml was obtained. The solution was stored in arefrigerator at −20° C. after being subpacked.

Sample to be tested: For each antibody of the present invention, 10 μland 2 μl of the culture supernatant of Expi293 cells expressing theantibody after transient transfection (the medium was Expi293 ExpressionMedium, Invitrogen, Catalog Number: A1435102; suspension culture wasperformed for 4 days at 100 rpm in a 8% CO2 incubator) were respectivelyadded to 990 μl and 998 μl of PBS to prepare antibody samples to betested of 1:100 and 1:500 dilutions.

Control sample: As negative control samples, the culture supernatant ofnormal cells (untransfected Expi293 cells; the medium was Expi293Expression Medium, Invitrogen, Catalog Number: A1435102; suspensionculture was performed for 4 days at 100 rpm in a 8% CO2 incubator) werealso diluted at 1:100 and 1:500.

2. Detection by ELISA

100 μl of 100 μg/ml sIL-4Rα solution was added to 9.90 ml of PBS, mixedup and down, and then an antigen coating buffer of 1.0 μg/ml wasobtained. The prepared antigen coating buffer was added to a 96-wellELISA plate (Corning) with a volume of 100 μl per well. The 96-wellELISA plate was incubated overnight in a refrigerator at 4° C. On thenext day, the solution therein was discarded, and PBS containing 2% BSAwas added to the 96-well ELISA plate row by row with a volume of 300 μlper well. The 96-well ELISA plate was incubated for 2 hours in arefrigerator at 4° C. Then the PBS containing 2% BSA was discarded andthe plate was washed 3 times with PBST. Diluted antibodies to be testedwere sequentially added to the corresponding wells, while the normalcell culture supernatants, as negative control samples, were added too.Three duplicate wells were made for each sample with a volume of 100 μlper well. The ELISA plate was wrapped with preservative film (orcovered) and incubated for 1 h at 10° C. in a constant temperatureincubator. Subsequently, the 96-well ELISA plate was taken out, and thesolution therein was discarded. After washing with PBST for 3 times, TMBsolution (Solarbio, Catalog Number: PR1200) was added to the 96-wellELISA plate row by row with a volume of 100 μl per well. The 96-wellELISA plate was placed at room temperature for 5 minutes, and 2 M H2SO4solution was added in immediately to terminate the reaction. The 96-wellELISA plate was placed in flexstation 3 (Molecular Devices), the valuesof OD450 were read, and the data were collected, calculated andanalyzed. Results were expressed relative to the affinity of controlantibody 1. See Tables 5a-5c below for the results.

TABLE 5a The affinities of the antibodies of the present invention forsIL-4Rα (antibodies whose affinities are significantly greater than thatof control antibody 1) Number of OD 450/ the antibodyOD450_(Control antibody 1) Control antibody 1 1 L1021H1000 2.42L1020H1000 2.27 L1019H1000 1.79 L1001H1000 1.56 L1012H1000 1.22L1000H1031 1.14 L1020H1031 1.12 L1000H1014 1.06 L1020H1029 1.01

TABLE 5b The affinities of the antibodies of the present invention forsIL-4Rα (antibodies whose affinities are equal to or slightly lower thanthat of control antibody 1) Number of OD 450/ the antibodyOD450_(Control antibody 1) L1010H1000 1 L1021H1029 1 L1011H1000 0.9L1008H1000 0.9 L1021H1031 0.9 L1024H1031 0.9 L1007H1000 0.8 L1020H10160.8 L1000H1029 0.8 L1000H1001 0.7

TABLE 5c The affinities of the antibodies of the present invention forsIL-4Rα (antibodies whose affinities significantly lower than that ofcontrol antibody 1) Number of OD 450/ the antibodyOD450_(Control antibody 1) L1024H1000 0.69 L1015H1000 0.67 L1000H10150.65 L1009H1000 0.64 L1021H1016 0.63 L1000H1023 0.63 L1000H1016 0.61L1000H1009 0.56 L1000H1032 0.53 L1017H1000 0.49 L1021H1007 0.44L1000H1027 0.44 L1020H1007 0.40 L1020H1027 0.40 L1012H1007 0.40L1000H1013 0.38 L1000H1007 0.37 L1000H1021 0.35 L1000H1028 0.33L1021H1027 0.29 L1016H1000 0.27 L1020H1020 0.26 L1000H1024 0.26L1021H1020 0.24 L1013H1000 0.24 L1024H1007 0.23 L1000H1020 0.23L1000H1035 0.22 L1000H1008 0.21 L1000H1025 0.21 L1000H1030 0.20L1000H1012 0.18 L1000H1022 0.18 L1022H1000 0.16 L1012H1031 0.16L1000H1017 0.13 L1000H1010 0.11 L1000H1026 0.11 L1012H1020 0.11L1012H1029 0.11 L1000H1036 0.10 L1000H1018 0.09 L1000H1034 0.09L1000H1011 0.09 L1000H1033 0.08 L1012H1016 0.08 L1018H1000 0.06L1023H1000 0.05 L1000H1038 0.04 L1000H1019 0.02 L1012H1027 0.02L1014H1000 0.01 L1000H1037 −0.01

Example 4: Pharmacokinetics of the Antibodies of the Present Inventionin Mouse a Series of Pharmacokinetic Experiments were Carried Out inMice to Further Screen Antibodies

6-8 week-old SPF Balb/c mice were selected and injected subcutaneouslywith antibodies (the antibodies of the present invention or controlantibody 2) in a dose of 5 mg/kg (weight of the mouse). Blood sampleswere collected at the time points before administration (0 h) and at 2,8, 24, 48, 72, 120, 168, 216, 264, 336 h after administration. For bloodsampling, the animals were anesthetized by inhaling isoflurane, bloodsamples were taken from the orbital venous plexus, and the samplingvolume for each animal was about 0.1 ml; 336 h after administration, theanimals were anesthetized by inhaling isoflurane and then euthanizedafter taking blood in the inferior vena cava.

No anticoagulant was added to the blood samples, and serum was isolatedfrom each sample by centrifugation at 1500 g for 10 min at roomtemperature within 2 h after blood sampling. The collected supernatantswere immediately transferred to new labeled centrifuge tubes and thenstored at −70° C. for temporary storage. The concentrations of theantibodies in the mice were determined by ELISA:

1. Preparation of Reagents

sIL-4Rα (PEPRO TECH, Catalog Number: 200-04R) solution: sIL-4Rα wastaken and 1 ml ddH2O was added therein, mixed up and down, and then asolution of 100 μg/ml was obtained. The solution was stored in arefrigerator at −20° C. after being subpacked.

Sample to be tested: 1 μl of serum collected at different time pointswas added to 999 μl of PBS containing 1% BSA to prepare a serum sampleto be tested of 1:1000 dilution.

Standard sample: The antibody to be tested was diluted to 0.1 μg/ml withPBS containing 1% BSA and 0.1% normal animal serum (Beyotime, CatalogNumber: ST023). Afterwards, 200, 400, 600, 800, 900, 950, 990 and 1000μl of PBS containing 1% BSA and 0.1% normal animal serum wererespectively added to 800, 600, 400, 200, 100, 50, 10 and 0 μl of 0.1μg/ml antibodies to be tested, and thus standard samples of theantibodies of the present invention were prepared with a finalconcentration of 80, 60, 40, 20, 10, 5, 1, or 0 ng/ml respectively.

2. Detection by ELISA

250 μl of 100 μg/ml sIL-4Rα solution was added to 9.75 ml of PBS, mixedup and down, and then an antigen coating buffer of 2.5 μg/ml wasobtained. The prepared antigen coating buffer was added to a 96-wellELISA plate (Corning) with a volume of 100 μl per well. The 96-wellELISA plate was incubated overnight in a refrigerator at 4° C. afterbeing wrapped with preservative film (or covered). On the next day, the96-well ELISA plate was taken out and the solution therein wasdiscarded, and PBS containing 2% BSA was added thereto with a volume of300 μl per well. The 96-well ELISA plate was incubated for 2 hours in arefrigerator at 4° C. after being wrapped with preservative film (orcovered). Then the 96-well ELISA plate was taken out and the solutiontherein was discarded, and the plate was washed 3 times with PBST. Thediluted standard antibodies and the sera to be detected weresequentially added to the corresponding wells, and three duplicate wellswere made for each sample with a volume of 100 μl per well. The ELISAplate was wrapped with preservative film (or covered) and incubated for1 h at room temperature. Subsequently, the solution in the 96-well ELISAplate was discarded and then the plate was washed with PBST for 3 times.Later, TMB solution (Solarbio, Catalog Number: PR1200) was added to the96-well ELISA plate row by row with a volume of 100 μl per well. The96-well ELISA plate was placed at room temperature for 5 minutes, and 2M H2SO4 solution was added in immediately to terminate the reaction. The96-well ELISA plate was then placed in flexstation 3 (MolecularDevices), the values of OD450 were read, the data were collected and theresults were calculated with Winnonlin software. The pharmacokineticresults were shown in FIG. 1 and Table 6 below.

TABLE 6 Pharmacokinetic results of the antibodies of the presentinvention in mouse Area Time Under the Half to Peak drug-time Volume ofClearance life peak concentration Curve distribution rate Number h hμg/ml h*μg/ml ml/kg ml/h/kg L1020H1031 Mean 269.34 72 33.79 7679.28138.92 0.38 value Standard 105.73 0.00 0.42 163.91 22.48 0.09 deviationL1012H1031 Mean 167.27 48 45.5 9852.3 91.3 0.38 value Standard 8.52 0.001.86 448.34 5.58 0.00 deviation Control Mean 56.67 36 7.88 1132.68288.92 3.79 antibody 2 value Standard 25.84 16.97 0.25 94.42 49.45 1.12deviation

Example 5: Pharmacokinetics of the Antibodies of the Present Inventionin Macaca fascicularis

A series of pharmacokinetic experiments were carried out in Macacafascicularises to further screen antibodies.

3-5 year-old Macaca fascicularises each weighting 2-5 Kg were selectedand injected subcutaneously with antibodies (the antibodies of thepresent invention or control antibody 2) in a dose of 5 mg/kg (weight ofthe Macaca fascicularis). The antibody or control antibody 2 to beadministered was accurately extracted with a disposable asepticinjector, and multi-point injections were made subcutaneously on theinner side of the thigh of the animal, and the injection volume perpoint was not more than 2 ml. Whole blood samples were collected fromthe subcutaneous vein of the hind limb of the animal at the time pointsbefore administration (0 h) and at 0.5, 2, 4, 8, 24, 48, 72, 120, 168,240, 336 h, 432 h, 504 h, 600 h, 672 h after administration. The bloodvolume collected from each animal was about 0.1 ml each time.

No anticoagulant was added to the blood samples, and serum was isolatedfrom each sample by centrifugation at 1500 g for 10 min at roomtemperature within 2 h after blood sampling. The collected supernatantswere immediately transferred to new labeled centrifuge tubes and thenstored at −70° C. for temporary storage. The concentrations of theantibodies in the Macaca fascicularises were determined according themethod as described in Example 4. The pharmacokinetic results are shownin FIG. 2 and Table 7 below.

TABLE 7 Pharmacokinetic results of the antibodies of the presentinvention in macaca fascicularis Area Time Under the Half to Peakdrug-time Volume of Clearance life peak concentration Curve distributionrate Number h h μg/ml h*μg/ml ml/kg ml/h/kg L1020H1031 Mean 254.95 48.0089.65 22189.91 75.94 0.22 value Standard 44.57 33.94 44.29 8557.15 22.950.10 deviation L1012H1031 Mean 185.75 48 65 16185.73 73.41 0.28 valueStandard 42.54 33.94 4.5 2506.98 0.81 0.06 deviation Control Mean 37.0316 37.82 2773.21 93.97 1.78 antibody 2 value Standard 18.03 11.31 6.75155.84 42.47 0.07 deviation

Example 6: Detection of Binding of the Antibodies of the PresentInvention to TF-1 Cells by FACS

1. Cell Culture

TF-1 cells (ATCC: CRL-2003™) frozen in liquid nitrogen were taken out,shaken in a 37° C. water bath gently to dissolve quickly. The dissolvedcell suspension was transferred to a 15 ml centrifuge tube, and then1640 medium (Hyclone, Catalog Number: SH30809.01B) was added therein to10 ml. The tube was centrifuged at 800 rpm for 5 min, the supernatanttherein was aspirated off, and the cell pellets were retained and washedonce again. The cell density was adjusted to 1×10⁵-1×10⁶ cells/ml with1640 medium containing 10% FBS (Hyclone, Catalog Number: SV30184.02) and2 ng/ml GM-CSF (Sino Biological, Catalog Number: 10015-H01H). Thesuspension was transferred to a T75 cell culture flask (Nunc), and thecells were statically cultured in an incubator (Thermo) at 37° C., 5%CO₂. Every 2-3 days, the cell suspension was taken out, centrifuged at800 rpm for 5 min, and the cells were resuspended in 10 ml of medium.Subsequently, 1×10⁶ cells were counted and transferred into a new T75cell culture flask, and the medium was supplemented thereto to 10 ml.The cells were continuously passaged for 2-3 times to reach a good state(the cells were bright, and had a slightly irregular shape whensuspended individually) for experiment.

2. Cell Treatment

Tf-1 cells were taken and counted under a microscope. The cells weredivided into three groups and placed into three 1.5 ml centrifuge tubesrespectively. The number of cells for each group was 1×10⁶. The cellswere centrifuged at 800 rpm for 5 min, and then resuspended in 1 ml ofcold PBS containing 1% BSA for washing once again. Subsequently, thecells were centrifuged at 800 rpm for 5 min, and 45 μl of cold PBScontaining 1% BSA was added to each centrifuge tube to resuspend thecells, and then 5 μl (500 μg/ml) of the present antibody L1021H1031,L1020H1031 or L1012H1031 was added to the first centrifuge tube, and 5μl of PBS was added to the second centrifuge tube as a negative control.The cells were allowed to stand on ice for 45 min and then centrifugedat 800 rpm for 5 min. Afterwards, the cells were resuspended in 1 ml ofcold PBS containing 1% BSA for washing once again, and centrifuged at800 rpm for 5 min. 499 μl of cold PBS containing 1% BSA was added toeach centrifuge tube to resuspend the cells, and then 1 μl ofFITC-labeled goat anti-human IgG (H+L) (Beyotime, Catalog Number: A0556)was added thereto. After standing on ice for 45 min, the cells werecentrifuged at 800 rpm for 5 min, and resuspended in 1 ml of cold PBScontaining 1% BSA for washing once again. Thus, the cells to be detectedwere obtained.

3. Detection by FACS

FACS instrument and its operating system were run according to correctprotocol. After correct parameters were set, the cells were added intothe detection tube to detect fluorescence signal of FL1 channel. Theresults were analyzed with FlowJo 7.6 software and are shown in FIG. 3 ,panels 3A-3C. It can be seen that the antibodies of the presentinvention are capable of specifically binding to TF-1 cells expressingIL-4Rα.

Example 7: Detection of the Blocking Effect of Soluble hIL-4 Rα(sIL-4Rα) on Binding of Antibodies to TF-1 Cells by FACS

1. Preparation of Reagents

The hIL-4 (Invivogen, Catalog Number: rhil-4) solution was prepared asdescribed in Example 2.

2. Cell Culture

The TF-1 cells (ATCC: CRL-2003™) were cultured as described in Example6.

3. Mixing of sIL-4Rα with the Antibodies of the Present Invention

10 μl (100 μg/ml) of sIL-4Rα and 5 μl (500 μg/ml) of antibodyL1021H1031, L1020H1031 or L1012H1031 of the present invention wereuniformly mixed in a 1.5 ml centrifuge tube at a molar ratio of 2:1. Themixture of 10 μl PBS and 5 μl of antibody L1021H1031, L1020H1031 orL1012H1031 of the present invention was served as a positive control and15 μl PBS was served as a negative control. The centrifuge tubes wereplaced in a 37° C. incubator for 1 h. 4. Cell treatment

TF-1 cells were taken and then counted under a microscope. The cellswere divided into four groups and placed into four 1.5 ml centrifugetubes respectively. The number of cells for each group was 1×10⁶. Thecells were centrifuged at 800 rpm for 5 min, and then resuspended in 1ml of cold PBS containing 1% BSA for washing once again. Subsequently,the cells were centrifuged at 800 rpm for 5 min, and 35 μl of cold PBScontaining 1% BSA was added to each centrifuge tube to resuspend thecells, and then 15 μl of each of different antibody mixtures and thecontrol prepared in step 3 was added to the tubes respectively. Afterstanding on ice for 45 min, the cells were centrifuged at 800 rpm for 5min, and resuspended in 1 ml of cold PBS containing 1% BSA for washingonce again, and centrifuged at 800 rpm for 5 min. 499 μl of cold PBScontaining 1% BSA was added to each centrifuge tube to resuspend thecells, and then 1 μl of FITC-labeled goat anti-human IgG (H+L)(Beyotime, Catalog Number: A0556) was added thereto. After standing onice for 45 min, the cells were centrifuged at 800 rpm for 5 min, andresuspended in 1 ml of cold PBS containing 1% BSA for washing onceagain. Thus, the cells to be detected were obtained.

5. Detection by FACS

FACS instrument and its operating system were run according to correctprotocol. After correct parameters were set, the cells were added intothe detection tube to detect fluorescence signal of FL1 channel. Theresults were analyzed with FlowJo 7.6 software and are shown in FIG. 4 ,panels 4A-4D. It can be seen that sIL-4Rα can specifically andeffectively block the specific binding of the antibodies of the presentinvention to TF-1 cells.

Example 8: Determination of Antibody Affinity by Bicore

An anti-human Fc (AHC) antibody (GE Healthcare) was coupled to a CM5chip, and then the antibody of the present invention was captured by AHCrespectively. Subsequently, different concentrations of human sIL-4Rαwere flowed through the surface of the chip on which the antibody of thepresent invention was captured. For capture and binding, the presentantibody was diluted to 2 μg/ml and the antigen sIL-4Rα was diluted to0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25.0, 50.0 and 100.0 nM. Theexperimental method was then established in the Biacore T200 controlsoftware, after which the experimental program was run for detection.The results are shown in Table 8 below.

TABLE 8 Detection results of the antibody affinity Ka Kd KD Number (×10⁵M⁻¹s⁻¹) (×10⁻⁵ s⁻¹) (10⁻¹⁰ M) Control antibody 1 7.63 52.3 6.85 Controlantibody 2 3.85 1.73 0.448 L1020H1031 4.61 4.79 1.04 L1012H1031 5.189.49 1.83

Example 9: Investigation of the Inhibitory Effect of the Antibodies ofthe Present Invention on TARC and MDC Release by ELISA

10 ml of fresh blood (donated) anticoagulated with heparin was mixedwith PBS at room temperature in a ratio of 1:1, and then the mixture wascarefully added to 20 ml of human lymphocyte separation solution(Solarbio, Catalog Number: P8610) prepared in advance. Aftercentrifuging at 1500 rpm for 30 min at room temperature, the PBMC layerwas carefully aspirated and washed twice with PBS. The cells werefinally diluted with 1640 medium containing 10% FBS and 200 IU/ml IL-2,and then added to a 24-well plate with 1×10⁶ cells per well. Afterwards,antibody L1020H1031 with a final concentration of 1000, 300, 100, 30 or10 ng/ml was added to each well, following by the addition of IL-4 witha final concentration of 10 ng/ml (or IL-13 with a final concentrationof 100 ng/ml) thereto. Detection was carried out according to the methodprovided with Human TARC ELISA kit (Abcam, Catalog Number: ab183366) orHuman MDC ELISA kit (Abcam, Catalog Number: ab179885) after 72 hours ofculture.

The results are shown in FIG. 5 , panels 5A-5B, showing that theantibodies of the present invention can effectively inhibit TARC and MDCrelease, and the inhibitory effect on release increases with theincreasing of antibody concentration.

Example 10: Effect of Specific Amino Acids on the Pharmacokinetics andExpression Levels of the Antibodies of the Invention

In vivo pharmacokinetics of the antibodies of the invention are furtherdetected and compared in this Example, in order to investigate thepossible effects of specific amino acids at specific positions on thepharmacokinetics of the antibodies in animals. The specific experimentalmethod was the same as that described in Example 4, and the results areshown in Table 9 below.

TABLE 9 Detection results of in vivo pharmacokinetics of the antibodiesof the present invention Area Time Under the Half to Peak drug-timeVolume of Clearance life peak concentration Curve distribution rate h hug/ml h*ug/ml ml/kg ml/h/kg L1020H1031 Mean 185.49 40 38.94 8188.8114.28 0.43 value Standard 18.52 13.86 2.33 510.47 6.5 0.05 deviationL1012H1001 Mean 161.26 48.00 12.36 2491.19 332.79 1.47 value Standard54.30 0.00 2.26 165.16 76.91 0.20 deviation L1001H1031 Mean 171.41 56.0042.74 9273.73 99.17 0.40 value Standard 6.12 13.86 7.38 1868.66 18.690.07 deviation L1020H1001 Mean 89.00 64.00 20.11 3481.40 164.14 1.30value Standard 16.70 13.86 2.14 268.39 22.86 0.20 deviation

From the specific sequence, the amino acid at position 103 in thesequence of the heavy chain H1031 (SEQ ID NO. 91) of the antibody (inCDR3) is Asp (103Asp), and the amino acid at position 104 is Tyr(104Tyr). Compared with antibodies that have no 103Asp and 104Tyr inheavy chain, the present antibodies which have 103Asp and 104Tyr have a2- to 4-fold higher area under the drug-time curve and an about 70%reduced clearance rate.

The expression levels of the antibodies of the present invention arealso detected and compared, in order to investigate the possible effectsof specific amino acids at specific positions on the expression of theantibodies. Culture and transfection of Expi293 cells were conductedaccording to Example 1, and the collected culture supernatant was thenpassed through a 0.22 μm filter and then purified by GE MabSelect Sure(Catalog Number: 11003494) Protein A affinity chromatography column inthe purification system GE AKTA purifier 10. The purified antibody wascollected and concentrated using Amicon ultrafiltration concentratingtube (Catalog Number: UFC903096) and then quantified. The quantitativeresults are shown in Table 10 below.

TABLE 10 Detection results of the expression levels of the antibodies ofthe present invention Expression level Antibody (×10⁻² mg/ml culturemedium) L1020H1031 8.39 L1001H1031 1.79 L1020H1001 4.04 L1012H1001 5.00L1023H1001 4.63 L1001H1001 1.75

From the specific sequence, the amino acid at position 31 in thesequence of the light chain L1012 (SEQ ID NO. 44), L1020 (SEQ ID NO. 55)or L1023 (SEQ ID NO. 51) of the antibody (in CDR1) is Ser (31Ser).Compared with antibodies that have no 31Ser in light chain, the presentantibodies which have 31Ser have a 2- to 5-fold higher expression level.

The above description for the embodiments of the present invention isnot intended to limit the present invention, and those skilled in theart can make various changes and variations according to the presentinvention, which are within the protection scope of the claims of thepresent invention without departing from the spirit of the same.

The invention claimed is:
 1. A method of treating or amelioratinginflammation or allergic disease in a subject in need thereof,comprising administering to the subject an isolated anti-interleukin 4receptor (IL-4R) antibody comprising a heavy chain variable domain (VH)comprising three heavy chain complementarity determining regions (CDRs)(CDR-H1, CDR-H2, and CDR-H3), and a light chain variable domain (VL)comprising three light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) wherein:a. the CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:2, the CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:3, the CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:8, the CDR-H1 comprises the amino acid sequence set forth in SEQ ID NO:14, the CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:18, and the CDR-H3 comprises the amino acid sequence set forth in SEQ IDNO: 19; b. the CDR-L1 comprises the amino acid sequence set forth in SEQID NO: 2, the CDR-L2 comprises the amino acid sequence set forth in SEQID NO: 4, the CDR-L3 comprises the amino acid sequence set forth in SEQID NO: 8, the CDR-H1 comprises the amino acid sequence set forth in SEQID NO: 14, the CDR-H2 comprises the amino acid sequence set forth in SEQID NO: 18, and the CDR-H3 comprises the amino acid sequence set forth inSEQ ID NO: 19; or c. the CDR-L1 comprises the amino acid sequence setforth in SEQ ID NO: 2, the CDR-L2 comprises the amino acid sequence setforth in SEQ ID NO: 4, the CDR-L3 comprises the amino acid sequence setforth in SEQ ID NO: 5, the CDR-H1 comprises the amino acid sequence setforth in SEQ ID NO: 14, the CDR-H2 comprises the amino acid sequence setforth in SEQ ID NO: 18, and the CDR-H3 comprises the amino acid sequenceset forth in SEQ ID NO:
 19. 2. The method of claim 1, wherein: a. the VLcomprises the amino acid sequence set forth in SEQ ID NO: 55 and the VHcomprises the amino acid sequence set forth in SEQ ID NO: 91; b. the VLcomprises the amino acid sequence set forth in SEQ ID NO: 54 and the VHcomprises the amino acid sequence set forth in SEQ ID NO: 91; or c. theVL comprises the amino acid sequence set forth in SEQ ID NO: 44 and theVH chain scqucncc comprises the amino acid sequence set forth in SEQ IDNO:
 91. 3. The method of claim 1, wherein the isolated antibody binds tohuman IL-4Rα.
 4. The method of claim 1, wherein the isolated antibodybinds to soluble human IL-4Rα.
 5. The method of claim 1, wherein theisolated antibody is a monoclonal antibody.
 6. The method of claim 1,wherein the isolated antibody is an immunoglobulin having a humanisotype of IgA, IgD, IgE, IgG or IgM.
 7. The method of claim 1, whereinthe isolated antibody is a human IgG1, IgG2, IgG3, or IgG4 subtype. 8.The method of claim 1, wherein the isolated antibody is a human IgG4subtype.
 9. The method of claim 1, wherein the inflammation or allergicdisease comprises atopic dermatitis.
 10. The method of claim 1, whereinthe inflammation or allergic disease comprises asthma.
 11. The method ofclaim 1, wherein the CDR-L1 comprises the amino acid sequence set forthin SEQ ID NO: 2, the CDR-L2 comprises the amino acid sequence set forthin SEQ ID NO: 3, the CDR-L3 comprises the amino acid sequence set forthin SEQ ID NO: 8, the CDR-H1 comprises the amino acid sequence set forthin SEQ ID NO: 14, the CDR-H2 comprises the amino acid sequence set forthin SEQ ID NO: 18, and the CDR-H3 comprises the amino acid sequence setforth in SEQ ID NO:
 19. 12. The method of claim 11, wherein the isolatedantibody is a monoclonal antibody.
 13. The method of claim 11, whereinthe inflammation or allergic disease is selected from the groupconsisting of allergic dermatitis, asthma, eosinophilic esophagitis,eczema, allergic rhinitis, nasal polyp, and rheumatoid arthritis. 14.The method of claim 11, wherein the inflammation or allergic disease isan autoimmune disease.
 15. The method of claim 11, wherein theinflammation or allergic disease is atopic dermatitis.
 16. The method ofclaim 11, wherein the inflammation or allergic disease is asthma. 17.The method of claim 11, wherein the isolated antibody is a human IgG4subtype.
 18. The method of claim 1, wherein the CDR-L1 comprises theamino acid sequence set forth in SEQ ID NO: 2, the CDR-L2 comprises theamino acid sequence set forth in SEQ ID NO: 4, the CDR-L3 comprises theamino acid sequence set forth in SEQ ID NO: 8, the CDR-H1 comprises theamino acid sequence set forth in SEQ ID NO: 14, the CDR-H2 comprises theamino acid sequence set forth in SEQ ID NO: 18, and the CDR-H3 comprisesthe amino acid sequence set forth in SEQ ID NO:
 19. 19. The method ofclaim 1, wherein the CDR-L1 comprises the amino acid sequence set forthin SEQ ID NO: 2, the CDR-L2 comprises the amino acid sequence set forthin SEQ ID NO: 4, the CDR-L3 comprises the amino acid sequence set forthin SEQ ID NO: 5, the CDR-H1 comprises the amino acid sequence set forthin SEQ ID NO: 14, the CDR-H2 comprises the amino acid sequence set forthin SEQ ID NO: 18, and the CDR-H3 comprises the amino acid sequence setforth in SEQ ID NO:
 19. 20. The method of claim 1, wherein the VLcomprises the amino acid sequence set forth in SEQ ID NO: 55 and the VHcomprises the amino acid sequence set forth in SEQ ID NO:
 91. 21. Themethod of claim 20, wherein the isolated antibody is a monoclonalantibody.
 22. The method of claim 20, wherein the inflammation orallergic disease is an autoimmune disease.
 23. The method of claim 20,wherein the inflammation or allergic disease is selected from the groupconsisting of allergic dermatitis, asthma, eosinophilic esophagitis,eczema, allergic rhinitis, nasal polyp, and rheumatoid arthritis. 24.The method of claim 20, wherein the inflammation or allergic disease isatopic dermatitis.
 25. The method of claim 20, wherein the inflammationor allergic disease is asthma.
 26. The method of claim 20, wherein theisolated antibody is a human IgG4 subtype.
 27. The method of claim 1,wherein the VL comprises the amino acid sequence set forth in SEQ ID NO:54 and the VH comprises the amino acid sequence set forth in SEQ ID NO:91.
 28. The method of claim 1, wherein the VL comprises the amino acidsequence set forth in SEQ ID NO: 44 and the VH comprises the amino acidsequence set forth in SEQ ID NO:
 91. 29. The method of claim 1, whereinthe inflammation or allergic disease is an autoimmune disease.
 30. Themethod of claim 1, wherein the inflammation or allergic disease isselected from the group consisting of allergic dermatitis, asthma,eosinophilic esophagitis, eczema, allergic rhinitis, nasal polyp, andrheumatoid arthritis.
 31. The method of claim 1, wherein theinflammation or allergic disease is atopic dermatitis.
 32. The method ofclaim 1, wherein the inflammation or allergic disease is asthma.